Third Codon Position Research Articles

Mitochondrial resolution of a deep branch in the genealogical tree for perching birds

Animal mitochondrial DNA (mtDNA) is known to contain information about the genealogical relations among closely related species and is shown here to yield information about distant relations as well. Our results also draw attention to the need for caution in using third positions of codons for tree construction. This is evident from comparative studies of the cytochrome b gene in 13 species representing major groups within the order of perching birds (Passeriformes). Sequences of a 924 base-pair segment of this gene were obtained from each of these species via the polymerase chain reaction and a novel set of versatile primers. With a woodpecker sequence as an outgroup, trees that separate songbirds from other perching birds and resolve the ancient branch leading to songbirds were obtained utilizing the conservative first and second positions of codons. Analysis of positions within codons suggests that, for deep branches, the skewed base composition at the fast-changing third positions can result in phylogenetic disinformation, which conflicts with the information retained in the first and second positions. The mitochondrial tree shows broad concordance with that based on hybridization of nuclear DNA; however, parsimony and maximum likelihood methods suggest a close kinship between thrushes and Australian babblers, in agreement with the traditional morphological classification.

Nucleotide sequence and codon usage of the elongation factor Tu(EF‐Tu) gene from Mycoplasma pneumoniae

The Mycoplasma pneumoniae tuf gene, encoding the elongation factor protein Tu, was cloned and sequenced. The nucleotide sequence of the mycoplasmal gene showed about 60% homology to the sequences of tuf genes of other prokaryotes, yeast mitochondria and Euglena gracilis chloroplasts, and about 75% similarity was found when comparing the deduced amino acid sequences of the various Tu proteins. The relatively low G + C content (40%) of the M. pneumoniae DNA was reflected in a low G + C content (44.6%) of the tuf gene, and in a preferential use of adenine and uracil at the third position of codons, yet codon usage analysis revealed the presence of almost all of the codons of the genetic code in the mycoplasmal gene. Southern blot hybridization of digested DNAs of 11 Mollicutes species with the entire M. pneumoniae tuf gene and with its 5' part suggested the presence of one copy only of this gene in the representative species of the Mollicutes. In this respect, the Mollicutes resemble Gram-positive bacteria and differ from the Gram-negative bacteria, which carry two copies of the tuf gene.

The N-ras oncogene is activated in a human medulloblastoma cell line

Medulloblastoma is a malignant brain tumor of early childhood whose cells resemble the primitive neuroepithelial cells found normally in the developing nervous system. Medulloblastoma may be caused by mutational events affecting primitive neuroepithelial cells and preventing their differentiation into postmitotic neurons. The human ras genes, H-ras, K-ras and N-ras, are members of a family of proto-oncogenes that are targets for mutational changes that convert these normal genes into active, transforming oncogenes. Here we report than the N-ras oncogene is activated in the human medulloblastoma cell line TE 671 by a mutation at the third position of codon 61. A point mutation at this location corresponds to a substitution of histidine for glutamine in the N-ras gene product, p21. The oncogenic activation was shown by focus-formation in NIH 3T3 cell transfection assays. The location of the mutation was established using oligonucleotide hybridization assays enhanced through in vitro amplification of N-ras coding sequences using the Taq polymerase chain reaction. N-ras activation may be one of the mutational events that subvert normal neuroectodermal differentiation and lead to medulloblastoma in children.

The consensus sequence of ice nucleation proteins from Erwinia herbicola, Pseudomonas fluorescens and Pseudomonas syringae

The consensus sequence of three bacterial ice nucleation proteins was determined by extrapolation from the nucleotide (nt) sequences of three ice nucleation-encoding genes, iceE (presented here), inaW and inaZ. The three proteins possess considerable similarity, so that a preferred amino acid is shown in most positions of the consensus. The corresponding genes show considerable divergence in the third nt positions of synonymous codons, suggesting that the proteins' conserved features have been maintained by selection. Therefore, the consensus sequence is likely to represent the components of primary structure most important to the ice nucleation function.

A novel delta zero-thalassemia arising from a frameshift insertion, detected by direct sequencing of enzymatically amplified DNA.

We describe a novel mutation in the delta globin gene of a compound heterozygote for delta o thalassemia and a deletion type G gamma + (A gamma delta beta) zero thalassemia. The delta was amplified using the polymerase chain reaction (PCR), and the amplified material was used in a direct sequencing experiment. The nucleotide sequence of the mutant delta gene showed that the insertion of an extra nucleotide at the third position of codon 91 in the second exon, which gives rise to a premature stop codon at position 94, leads to the silencing of this gene. The presence of the mutation in the carriers of delta-thalassemia in this family was confirmed by dot blot hybridization. A possible model for the insertion of the extra nucleotide is discussed.

Reversion of trpA nonsense mutations by deletion of the chain-termination codons

This paper describes a novel mechanism for reversion of nonsense mutations in the trpA gene of Escherichia coli. This mechanism, deletion of the nonsense codon, was discovered in the course of selecting for missense revertants of trpA(UGA211) and for catalytically active tryptophan syntherase alpha chain revertants of trpA(UAA234) and trpA(UAG234). Each type of revertant trpA was cloned and its DNA sequence determined. trpA(UGA211) gave rise to two previously unidentified types of missense revertant. The first type was expected, namely trpA(CGA211), the result of a base substitution event. The other type, representing approximately 1% of the missense revertants, was unexpected on the basis of single base substitutions and an understanding of which amino acids are functional at alpha chain position 211. It was found to be the result of a 21 base-pair deletion of a region containing codon 211. The tryptophan-independent revertants of both position 234 nonsense mutants occurred at a frequency of approximately 2 per 10 9 viable cells. They were identical in that they both resulted from a 3 base-pair deletion, namely deletion of the chain-terminating codon at position 234. One of them, however, also displayed an A instead of the normal G in the third position of codon 235. The revertants were characterized according to growth in different media and tryptophan synthetase assays performed on crude extracts. These types of mutants should prove interesting and important for the elucidation of alpha chain structure-function relationships, for insight into the assembly and interaction of subunits in this model multienzyme complex, and for the study of mechanisms by which deletions can be generated.

Nucleotide Sequence of the Bacillus licheniformis Homologue of the Sporulation Locus spoIIA of Bacillus subtilis

The spoIIA locus of Bacillus licheniformis was cloned into the phage vector phi 105J9; selection was based on the ability of the clone to complement mutations in both the first and the last of the spoIIA genes of B. subtilis. The B. licheniformis DNA was subcloned into M13 and sequenced; it includes three genes of identical lengths to those of B. subtilis. The average interspecies difference in nucleotide sequence is 24%; C occurs at the third position of codons 55% more often in the B. licheniformis than in the B. subtilis sequence. The average difference in predicted amino acid sequence is 11%, but the distribution of these differences is far from random, and there are several long stretches of amino acid sequence that are identical in the two organisms. The distribution of non-conservative amino acid changes between the species is also strikingly non-random; no such changes are found in those regions in which missense mutations are known in B. subtilis. Each species has an open reading frame 5' of the spoIIAA gene, but the predicted amino acid sequence, and the distribution of differences between the two species in both nucleotides and predicted amino acids, suggest that these open reading frames are not expressed. Both species have regions 3' of the open reading frames which resemble rho-independent terminators of transcription, but that in B. licheniformis is longer and could form a more elaborate secondary structure than that in B. subtilis.

A novel lysine-substituted nucleoside in the first position of the anticodon of minor isoleucine tRNA from Escherichia coli.

A minor species of isoleucine tRNA (tRNA(minor Ile)) specific to the codon AUA has been isolated from Escherichia coli B and a modified nucleoside N+ has been found in the first position of the anticodon (Harada, F., and Nishimura, S. (1974) Biochemistry 13, 300-307). In the present study, tRNA(minor Ile)) was purified from E. coli A19, and nucleoside N+ was prepared, by high-performance liquid chromatography, in an amount (0.6) A260 units) sufficient for the determination of chemical structures. By 400 MHz 1H NMR analysis, nucleoside N+ was found to have a pyrimidine moiety and a lysine moiety, the epsilon amino group of which was involved in the linkage between these two moieties. From the NMR analysis together with mass spectrometry, the structure of nucleoside N+ was determined as 4-amino-2-(N6-lysino)-1-(beta-D-ribofuranosyl)pyrimidinium ("lysidine"), which was confirmed by chemical synthesis. Lysidine is a novel type of modified cytidine with a lysine moiety and has one positive charge. Probably because of such a unique structure, lysidine in the first position of anticodon recognizes adenosine but not guanosine in the third position of codon.

A novel lysine-substituted nucleoside in the first position of the anticodon of minor isoleucine tRNA from Escherichia coli.

A minor species of isoleucine tRNA (tRNA(minor Ile)) specific to the codon AUA has been isolated from Escherichia coli B and a modified nucleoside N+ has been found in the first position of the anticodon (Harada, F., and Nishimura, S. (1974) Biochemistry 13, 300-307). In the present study, tRNA(minor Ile)) was purified from E. coli A19, and nucleoside N+ was prepared, by high-performance liquid chromatography, in an amount (0.6) A260 units) sufficient for the determination of chemical structures. By 400 MHz 1H NMR analysis, nucleoside N+ was found to have a pyrimidine moiety and a lysine moiety, the epsilon amino group of which was involved in the linkage between these two moieties. From the NMR analysis together with mass spectrometry, the structure of nucleoside N+ was determined as 4-amino-2-(N6-lysino)-1-(beta-D-ribofuranosyl)pyrimidinium (lysidine), which was confirmed by chemical synthesis. Lysidine is a novel type of modified cytidine with a lysine moiety and has one positive charge. Probably because of such a unique structure, lysidine in the first position of anticodon recognizes adenosine but not guanosine in the third position of codon.

Directional mutation pressure and transfer RNA in choice of the third nucleotide of synonymous two-codon sets.

Bacterial species have diverged into a series of families, some with high G + C content in their DNA, and other with high A + T content, resulting, respectively, from G.C- and A.T-directional mutation pressures. Such mutation pressure (G.C/A.T pressure) may be an important determinant for codon usage. It has also been suggested that tRNA acts as a selective constraint for determining codon usage. We have studied the relation between G.C/A.T pressure and tRNA constraints in determining choice of the third nucleotide of eight two-codon sets, using codon usage data obtained from protein genes in four bacterial species, Mycoplasma capricolum, Bacillus subtilis, Escherichia coli, and Micrococcus luteus, and in liverwort (Marchantia polymorpha) chloroplasts. The genomic G + C contents of these range from 25% to 74%. The results demonstrate that tRNA levels act additively to A.T and G.C pressure in affecting contents of A (pairing with *UNN anticodons, in which *U indicates a 2-thiouridine derivative) and C (pairing with GNN anticodons) or G (pairing with CNN anticodons), respectively, in third nucleotide positions of codons.

Cloning genes for the biosynthesis of a macrolide antibiotic.

Macrocin-O-methyltransferase (MacOMeTase) catalyzes the final enzymatic step in the biosynthesis of tylosin in Streptomyces fradiae. A 44-base mixed oligonucleotide probe containing only guanosine and cytidine in the third position of degenerate codons was synthesized based on the amino acid sequence of the amino terminus of MacOMeTase. Plaque blot hybridization to a bacteriophage lambda library and colony blot hybridization to a cosmid library of S. fradiae DNA identified recombinants that contained overlapping fragments of chromosomal DNA. The nucleotide sequence of the cloned DNA verified that the DNA contained the coding sequence for MacOMeTase. Recombinant plasmids transformed mutants blocked in tylosin biosynthesis and complemented tylF (the structural gene for MacOMeTase) and tyl mutations of eight other classes.

Transitions, transversions, and the molecular evolutionary clock.

Nucleotide substitutions in the form of transitions (purine-purine or pyrimidine-pyrimidine interchanges) and transversions (purine-pyrimidine interchanges) occur during evolution and may be compiled by aligning the sequences of homologous genes. Referring to the genetic code tables, silent transitions take place in third positions of codons in family boxes and two-codon sets. Silent transversions in third positions occur only in family boxes, except for A = C transversions between AGR and CGR arginine codons (R = A or G). Comparisons of several protein genes have been made, and various subclasses of transitional and transversional nucleotide substitutions have been compiled. Considerable variations occur among the relative proportions of transitions and transversions. Such variations could possibly be caused by mutator genes, favoring either transitions or, conversely, transversions, during DNA replication. At earlier stages of evolutionary divergence, transitions are usually more frequent, but there are exceptions. No indication was found that transversions usually originate from multiple substitutions in transitions.

Biosynthesis of reovirus-specified polypeptides: Molecular cDNA cloning and nucleotide sequence of the reovirus serotype 1 lang strain s2 mRNA which encodes the virion core polypeptide σ 2

Human reovirus serotype 1 Lang strain s2 mRNA, which encodes the virion inner capsid core polypeptide sigma 2, was cloned as a cDNA:mRNA heteroduplex in Escherichia coli using phage M13. A complete consensus nucleotide sequence was determined. The Lang strain s2 mRNA is 1331 nucleotides in length and possesses an open reading frame with a coding capacity of 335 amino acids, sufficient to account for a sigma 2 polypeptide of 37,682 daltons. Comparison of the serotype 1 Lang s2 sequence derived from cDNA clones of s2 mRNA with the serotype 3 Dearing S2 sequence derived from cDNA clones of the S2 dsRNA genome segment reveals 86 percent homology at the nucleotide level. The predicted sigma 2 polypeptides of the Lang and Dearing strains display 98 percent homology at the amino acid level. Of 147 silent nt differences in the translated region, 136 were in the third base position of codons.

Codon contexts in enterobacterial and coliphage genes.

This investigation of the codon context of enterobacteria, plasmid, and phage protein genes was based on a search for correlations between the presence of one base type at codon position III and the presence of another base type at some other position in adjacent codons. Enterobacterial genes were compared with eukaryotic sequences for codon context effects. In enterobacterial genes, base usage at codon position III is correlated with the third position of the upstream adjacent codon and with all three positions of the downstream codon. Plasmid genes are free of context biases. Phage genes are heterogeneous: MS2 codons have no biased context, whereas lambda genes partly follow the trends of the host bacterium, and T7 genes have biased codon contexts that differ from those of the host. It has been reported that two successive third-codon positions tend to be occupied by two purines or two pyrimidines in Escherichia coli genes of low expression level. Here, the extent to which highly expressed protein genes can modulate base usage at two successive codon positions III, given the constraints on codon usage and protein sequence that act on them, was quantified. This demonstrates that the above-mentioned favored patterns are not a characteristic of weakly expressed genes but occur in all genes in which codon context can vary appreciably. The correlation between successive third-codon positions is a distinct feature of enterobacteria and of some phages, one that may result from adaptation of gene structure to translational efficiency. Conversely, codon context in yeast and human genes is biased--but for reasons unrelated to translation.

Carcinogens with diverse mutagenic activities initiate neoplastic guinea pig cells that acquire the same N-ras point mutation.

N-ras has been identified by molecular cloning and DNA sequence analysis as the activated oncogene in carcinogen-induced guinea pig transformation. The deduced guinea pig amino acid sequence differs from that of human and mouse by 1 and 4 residues, respectively; the mismatches were in the C-terminal half of the fourth exon. The activated N-ras clone has an AT to TA transversion at the third position of codon 61 which results in the insertion of histidine instead of glutamine. The same activated N-ras gene with the identical mutation was found in all lines regardless of initiating carcinogen (aromatic aryl hydrocarbons or alkylating agents). These results suggest that the mutational event was independent of the mutagenic activity of the initiating carcinogen.

Tolerated variations in a genome: the case of closely related Bacillus phages PZA, φ29 and φ15 — a review

Restriction-site analysis was used to estimate the relationship of bacteriophages PZA, φ29 and φ15. Complete nucleotide sequences of PZA and φ29 genomes were compared and tolerated variations were assessed. Most of the base-pair changes are silent nucleotide substitutions in the third position of codons but amino acid changing substitutions are also observed. The terminal portions of the phage genomes diverged faster than their central parts. Gene mutations in phage PZA were induced by hydroxylamine and their frequency was compared with the evolutionary mutability.

Mosquito mitochondrial transfer RNAs for valine, glycine and glutamate: RNA and gene sequences and vicinal genome organization 19851230

We report the sequences of 3 transfer RNAs from mosquito (Aedes albopictus) mitochondria, those for valine (anticodon UAC), glutamic acid (anticodon UUC) and glycine (anticodon UCC), as well as sequences for the corresponding genes and for some neighboring mitochondrial genes. TRNAval is notable for its high level of psi, tRNAglu for its low level of G and C, and tRNAgly is notable in that it appears as two species widely separated in gel electrophoresis, differing only in modification status. TRNAglu is the first sequenced insect mitochondrial tRNA that would be expected to engage in U.R wobble (where U is a modified U in the first position of the anticodon, and R is G or A in the third position of codons), if the insect system followed the modified wobble rules proposed for mammalian and fungal mitochondria; and the sequence determined does fit the proposal. The gene for tRNAval follows immediately that for 12S ribosomal RNA. The gene for tRNAglu occurs in a cluster of 6 tRNA genes that is separated from the gene for tRNAgly by a short reading frame. Features of the DNA sequences are discussed with reference to Drosophila, and mammalian, mitochondrial genome organization.

Nucleotide sequence of the late region of Bacillus phage φ29 completes the 19285-bp sequence of φ29 genome. Comparison with the homologous sequence of phage PZA

The 12177-bp nucleotide (nt) sequence of the late region of Bacillus phage φ29 genome was determined. This sequence completes the entire 19285-bp sequence of phage φ29 DNA. Eleven open reading frames were found in this region, and these were assigned to eleven late genes. Ribosome-binding sites and a potential transcriptional promoter and terminator are considered. The nt sequence was compared to the homologous region of the closely related phage PZA and tolerated variations at the nt and amino acid (aa) level were evaluated. The most frequent changes are silent nt substitutions in the third position of codons, but aa substitutions are also found.

Third letters in codons counterbalance the (G + C)-content of their first and second letters

The correlation among the frequencies of appearance of G and C bases at the first, second and third positions of codons is examined. It is found that the redundancy at the wobbling third base is utilized to counter-balance the local (G + C)-content variation due to the first and second bases of codons, so that a homogeneous (G + C)-content is provided to each individual gene. We speculate that this levelling tendency of (G + C)-content comes from the functional constraint to achieve a uniform double-helix stability in a gene.

An alternative approach to deoxyoligonucleotides as hybridization probes by insertion of deoxyinosine at ambiguous codon positions.

Two deoxyoligonucleotide probes (23-mer and 26-mer) carrying deoxyinosine residues (I) at positions corresponding to ambiguous nucleotides derived from amino acid sequence have been synthesized by the phosphotriester method using a polymer support. The 23-mer and 26-mer corresponded to the mRNA for 8 amino acids from gastrin and 9 amino acids from cholecystokinin, respectively. The dIs have been used where the base in the third position of the amino acid codon is ambiguous. These deoxyoligonucleotides were used as probes for hybridization with colonies containing the corresponding cDNAs or genes. The hybrid formed between a gastrin clone and the 23-mer that harbors 5 dIs was dissociated at 50-55 degrees C, suggesting that deoxyinosine did not significantly effect the stabilization or destabilization of the DNA duplex. A similar result was obtained using the 26-mer that contains 5 dIs and a phage clone DNA of the cholecystokinin gene. Thus oligonucleotide probes with deoxyinosine residues at ambiguous points seem to be useful as hybridization probes for cloning genes for proteins containing amino acids with degenerate codons.